Apparatus and method for evaluating characteristics of target molecules

ABSTRACT

Arrangements are described for evaluating characteristics of target molecules. A biochip is received which includes a substrate to which charged probe molecules are attached. The probe molecules have a marker to allow generating signals indicative of the distance of a portion of the probe molecule from the substrate. The signals are detected and means for an external electric field is generated to which the probe molecules are exposed. A control means acts to: (A) apply an external electric field causing the portion of the probe molecule to approach the substrate, and (B) apply an external electric field causing the portion of the probe molecule to move away from the substrate. The signal is recorded as a function of time during step (A) and/or step (B). Steps (A) and (B) are repeated for a predetermined number of times and the recorded signals are combined.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/850,930, filed Mar. 26, 2013, which is a continuation of PatentCooperation Treaty Application PCT/EP2011/004833, filed Sep. 27, 2011,which in turn claimed priority from European Patent Application10180282.5, filed Sep. 27, 2010; each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of nanobiotechnology. Moreparticularly, the present invention relates to a surface-based moleculardynamics measurement concept that has been recently introduced by thepresent inventors.

BACKGROUND OF THE INVENTION AND RELATED PRIOR ART

In the field of nanobiotechnology, particular attention has beenfocussed on biochips, such as DNA chips and protein chips as aneffective means for simplifying nucleic acid and protein testing inareas such as clinical diagnosis and drug development. Biochips, whichare often also referred to as micro arrays, are substrates formed fromglass, silicon, plastic, metal or the like on which multiple differingprobes composed of bio molecules such as DNA and proteins are placed asspots in high-density areas. Binding of target molecules with probemolecules is traditionally detected by means of a fluorescence label orthe like associated with the target molecules.

The present inventors have recently introduced a chip-compatible schemefor the label-free detection of bio molecules by a surface-basedmolecular dynamics measurement, which the inventors termed “switchSENSEmethod” for reasons that will become apparent below. In this method, theprobe molecule is a charged molecule, particularly a charged polymer ora charged nanowire, that has a first portion attached to a substrate.The probe molecule has a marker allowing to generate signals indicativeof the distance of a second portion, e.g. the distal end of the probemolecule from the substrate. The probe molecule further has a capturepart capable of binding with certain target molecules that are to bedetected.

In the switchSENSE method, the probe molecule is subjected to anexternal AC field. Since the probe molecule is charged, depending on thecurrent polarity of the external field, said second portion of the probemolecule which is not directly attached to the substrate will approachor move away from the substrate. The change of configuration can bethought of as a switching between a “standing” configuration, in whichthe second portion is maximally removed from the substrate, and a“lying” configuration, in which the second portion is closest to thesubstrate. However, since the probe molecule is not limited to anyspecific shape, this terminology is rather metaphorical and should notbe understood to impose any restriction on the type or shape of theprobe molecule used.

By analyzing the switching behaviour between the standing and lyingconfigurations, it is possible to detect the presence of a targetmolecule bound to the probe molecule. Importantly, for this detection itis not necessary that the target molecule itself is labelled in anysense, which is why the switchSENSE method is referred to as a“label-free detection method”.

For a better illustration of the switchSENSE method, reference is madeto FIGS. 1 to 3, which have been taken from Ulrich Rant et. al.,“Detection and Size Analysis of Proteins with Switchable DNA Layers”,Nano Letters 2009, Vol. 9, Nr. 4, 1290-1295 (prior art document 1)co-authored by some of the present inventors, which is included into thepresent disclosure by reference. It is to be understood that referenceto this work is only meant to explain the relevant prior art but is notintended to limit the invention in any way.

In prior art document 1, (negatively charged) DNA molecules were used asprobe molecules. In particular, synthetic 72-mer oligonucleotides weremodified with a thiol (HS) to covalently tether the strands to thesubstrate, which was a gold surface in this prior art. The distal end ofthe DNA molecule was labelled with a fluorescent marker (cyanine die,Cy3). The single-stranded (ss) HS-ss DNA-Cy3 sequence was hybridizedwith a complementary strand that was modified with a protein binding tag(PBT), so that a double-stranded capture probe was formed.

The DNA layer thus formed was activated by an external electric field.Alternating potentials were applied in aqueous salt solution between thegold surface, acting as a work electrode, and a counter electrode. Theapplied bias polarizes the electrode interface, leading to the formationof Gouy-Chapman-Stern screening layer on the solution side. Theresulting electric field was confined to the electrode proximity, withan extension of only a few nanometers, but was very intense with a fieldstrength of up to 100 kV/cm even for low bias potentials of less than 1V. Since the DNA is intrinsically negatively charged along itsdeprotonized phosphate backbone, the molecules align in the electricfield and the DNA conformation can be switched between theabovementioned “standing” and “lying” state, depending on the polarityof the applied bias.

The switching action can be monitored by observing the fluorescence fromthe Cy3 fluorescence labels attached to the DNAs' upper ends. Anon-radiative energy transfer from the optically excited dye to surfaceplasmons in the gold electrode quenches the emitted fluorescenceintensity when the fluorescence marker, i.e. the upper DNA end,approaches the surface. Accordingly, the fluorescence marker is anexample of a marker allowing to generate signals indicative of thedistance of a second portion of the probe molecules (i.e. in thisexample, the distal ends) from the substrate, i.e. the gold surface.

In FIG. 1C, the probe molecule 10 used in prior art document 1 isschematically shown. As mentioned above, the probe molecule 10 is adouble-stranded DNA, having a fluorescence marker (dye) 12 and a proteinbinding tag 14 attached to its distal end. Further, the gold substrate16 and a voltage source 18 for biasing the gold surface 16 areschematically shown. If a negative voltage is applied to the goldsurface or work electrode 16, the probe molecule 10 is repelled andpushed to the standing configuration schematically shown in FIG. 1C.Conversely, if a positive voltage is applied to the gold substrate 16,the distal end of probe molecule 10 approaches the same, i.e. the probemolecule 10 acquires the lying configuration. As is schematicallyindicated in FIG. 1C, due to the stiffness of the molecule, theswitching between the standing and lying configurations is thought of asa rotation of the DNA around its fixed end.

With reference to FIGS. 1A-1D, and specifically FIG. 1B, the switchingbetween the standing and lying configurations can be detected byobserving the fluorescence light emitted by the marker 12. If the probemolecule 12 is in the standing configuration, the distance between thefluorescence marker 12 and the gold electrode 16 is the largest, and thefluorescence emission is not quenched. Accordingly, in this standingconfiguration, the detected fluorescence is the largest. Conversely, ifthe distal end of probe molecule 10 and hence the fluorescence marker 12approaches the gold electrode 16, the aforementioned non-radiativeenergy transfer from the optically excited fluorescence marker 12 tosurface plasmons in the gold electrode 16 quenches the emittedfluorescence intensity and thus leads to a decreased fluorescenceintensity, see FIG. 1B. Herein, the difference in fluorescence intensitybetween the standing and lying configurations is referred to as “ΔF”.

As is demonstrated in the above-referenced prior art document 1, it isshown that binding of target molecules (proteins in the specificexample) to the probe molecule 10 alters the attainable switchingamplitude of the probe molecule layer, which is most likely caused byinducing steric interactions between neighbouring molecules. Thisphenomenon can be observed in FIG. 1B, where at the time 600 s,unlabelled streptavidin (SA) is added to the fluid environment and bindsto the protein binding tag 14. As more and more of the SA binds with theprobe molecule 10, ΔF decreases, until it reaches a plateau atapproximately 1200 s. Accordingly, the modulation of ΔF serves as anindicator that a target molecule (in this case SA) has bound to theprotein binding tag 14 of the probe molecule 10. Further, by observingthe dynamic behaviour of ΔF, also the binding kinetics can bedetermined, in particular a binding or dissociation rate between thetarget and probe molecules, an affinity constant and a dissociationconstant.

As is further demonstrated in prior art document 1, this label-freedetection of a target molecules binding to the probe molecules via ΔF isextremely sensitive, with a detection limit below 100 fmol/l.

Further, the inventors of the present invention have shown that it ispossible to discern information about the size of the target molecule byanalyzing the frequency response of the switching dynamics, as will beexplained with reference to FIG. 2 A-B taken from prior art document 1as well. In FIG. 2A, the normalized switching amplitude ΔF as a functionof biasing voltage frequency is shown for the pristine probe DNA in theupper curve and after binding of the immunoglobulin G (IgG) antibody ofa sheep (lower curve). As a switching field, a sinusoidal ac biasvoltage is applied to the work electrode, the frequency of which isvaried. In FIG. 2A, the resulting frequency spectra are shown, whicheach comprise three distinct regimes:

-   (i) for low frequencies (<1 kHz), the DNA molecules follow the    electrical excitation with maximal efficiency, leading to maximum    oscillation of the probe molecule 10 and hence a maximum value of    ΔF;-   (ii) in an intermediate regime, the switching amplitude ΔF decays;    and-   (iii) in a high frequency region (>100 kHz), the probe molecule 10    cannot be driven by the applied AC potential anymore and hence ΔF    vanishes.

The frequency range (ii) is of particular interest because it reflectsthe finite time constant of the switching process. To compare theswitching dynamics of different samples, the frequency at which theamplitude ΔF has decreased to 50% of its initial value is evaluated. Thefrequency is called “cut-off frequency f_(c)” in the following. As canbe seen from FIG. 2, for the pristine DNA layer a cut-off frequencyf_(c,1)=18 kHz is found. After IgG (sheep) is bound to the DNA layer,one observes a pronounced decrease of the cut-off frequency, namelyf_(c,2)=11.5 kHz. Accordingly, it is seen that the binding of targetmolecules to the probe molecules slows down the dynamics of theswitching process down.

It is believed that the slowing down of the switching process is causedby the increased hydrodynamic drag due to the attached target molecule.It is further believed that the increment in molecular weight does notplay an as important role, since the DNA motion is extremelyover-damped, so that inertial effects can be neglected. As has beenshown by the inventors of the present invention, the frequency shiftinduced by target molecules can be used to determine the targetmolecules' size, in particular their effective Stokes radius. Withregard to the example of FIG. 2, the inventors have measured frequencyshifts for various antibodies and antibody fragments. FIGS. 3A and 3Bsummarize cut-off frequency measurements for proteins of varying size.The normalized cut-off frequency is plotted versus the normal molecularweight in panel A (Fab sheep (50 kDa), streptavidin (75 kDa), Fab2, goat(100 kDa), IgG sheep (150 kDa) and IgG goat (160 kDa)). The cut-offfrequency versus the respective hydrodynamic diameters Dh are shown inpanel B. FIG. 3C shows the D_(h) distribution measured by dynamic lightscattering, see prior art document 1 for more details.

As can be seen from FIG. 3, a monotonous decrease of the normalizedcut-off frequency is found for increasing size of the bound protein. Inparticular, Fab and Fab2 fragments can be clearly discriminated fromuncleaved antibodies. Accordingly, measuring the frequency response ofthe fluorescence amplitude, the size of the target molecules can beevaluated with remarkable precision.

As is obvious from the above, using the switchSENSE method, both thebinding of the target molecule as such as well as the size of the targetmolecules can be evaluated for unlabelled targets. In particular, theevaluation of target molecule size by analyzing the frequency responsehas proven to be a very powerful tool that requires only limitedexperimental effort and proved to be very robust. The frequency responseanalysis is also the subject of further publications and patentapplications co-authored by some of the present inventors, see inparticular U. Rant et. al, “Switchable DNA Interfaces for the HighlySensitive Detection of Label-free DNA Targets”, PNAS (270), Vol. 104,Nr. 44, p. 17364-17369, EP 2 192 401 A1 and US 2005/0069932 A1.

While the combined detection and size evaluation of target moleculesaccording to the above prior art has proven to be very successful, thereis an on-going desire to increase the precision and reliability of theevaluation of characteristics of the target molecules. A further objectof the invention is to provide a method for evaluating characteristicsof a target molecule that would be particularly suitable forimplementing in commercially available apparatuses, thus lifting theswitchSENSE technology from a scientific concept to a practical toolthat can be routinely used not only in academic research but also inpharma and biotech industry as well as laboratories for clinicaldiagnostics and hospitals. A further problem underlying the invention isto provide an apparatus that would allow evaluating characteristics of atarget molecule bound to a probe molecule with good precision, yieldingreliable and trustworthy results to routine users who cannot be expectedto question the analysis results but instead need to rely on them.

SUMMARY OF THE INVENTION

The above objects are met by an apparatus for evaluating one or morecharacteristics of target molecules according to claim 1 and by a methodaccording to claim 8. Advantageous further developments are defined inthe dependent claims.

According to a first aspect of the invention, an apparatus forevaluating one or more characteristics of target molecules is provided.The apparatus of the invention comprises means for receiving a biochip,wherein the biochip comprises a substrate to which probe molecules areattached with a first portion thereof. The probe molecules are chargedand have a marker for allowing to generate signals indicative of thedistance of a second portion of the probe molecule from the substrate.Herein, the substrate may be a work electrode and the marker could be afluorescence marker as in the prior art described above, but theinvention is not limited to this.

Further, the apparatus of the invention comprises means for detectingthe signal generated with the marker and means for a generating anexternal electric field which the probe molecules are exposed to whenthe biochip is received in the receiving means. Also, the apparatus ofthe invention comprises a control means configured to control theelectric field generating means to

-   (A) apply an external electric field causing the second portion of    the probe molecule to approach the substrate when the biochip is    received in the receiving means, and-   (B) apply an external electric field causing the second portion of    the probe molecule to move away from the substrate.

Herein, the control means is further configured to control the signaldetecting means to record the signal indicative of the distance of thesecond portion from the substrate as a function of time during step (A)and/or step (B). The control means is further configured to control theelectric field generation means and the detecting means such as torepeat steps (A) and (B) for a predetermined number of times and isconfigured to combine the recorded signals such as to generate anaveraged time-resolved signal indicative of the process of the secondpart of the probe molecule approaching and/or moving away from thesubstrate.

Finally, the apparatus comprises an analysis module for analyzing and/orprocessing said combined signal such as to determine said one or morecharacteristics of said target molecule, and preferable an output devicefor outputting the at least one or more characteristics of said targetmolecule. Alternatively, the apparatus may comprise an interfaceallowing to couple the apparatus directly or indirectly with an outputdevice such as a display.

Unlike the prior art discussed above, the apparatus of the inventiondispenses with the above concept of determining the size or effectiveStokes radius of the target molecules by the frequency response of theswitching amplitude ΔF. Instead, the apparatus of the invention carriesout a time-resolved measurement of the switching process itself, i.e. ofthe transition between the standing configuration and the lyingconfiguration and vice versa.

In spite of the indisputable success of the characterization viafrequency response, the inventors have found out that the reliability ofthe evaluation of target characteristics can be improved by replacingthe frequency response measurement, which conceptually is a spectralapproach, by a time-resolved measurement. In fact, the inventors havefound out that while the frequency response measurement in somescenarios is very sensitive when it comes to evaluating the size oftarget molecules, in other scenarios it will fail to distinguish targetmolecules of different sizes, which can however be distinguished withthe apparatus and method of the invention based on a time-resolvedmeasurement of the switching process itself. From the inventors' pointof view, who have developed and explored the frequency response methodthemselves, this is a surprising and unforeseeable result.

It should be noted that one of the inventors had presented time-resolvedfluorescence measurements of single- and double-stranded DNA tethered toa gold surface in an earlier publication (see U. Rant et al.,“Dissimilar Kinetic Behaviour of Electrically Manipulated Single- andDouble-Stranded DNA Tethered to a Gold Surface”, Biophysical Journal,Vol. 90 (2006), p. 3666-3671). However, this time-resolved measurementwas only related to the DNA as such, not to a DNA functionalized as aprobe molecule, and in particular not a probe molecule to which a targetmolecule was or could have been bound. In other words, this prior workwas not related to the characterization of target molecules.

What is more, the experience the inventor gained with this prior workwould not at all have suggested to employ a time-dependent measurementof the switching process for evaluating characteristics of targetmolecules bound to probe molecules. Namely, using the box carmeasurement approach in this prior work, recording a singletime-resolved measurement of one switching cycle took several days,which is of course prohibitive for any application in target analysis,where results are needed quickly and where the targets will only bind tothe probe molecule for a limited time. Surprisingly, however, theinventors could confirm that it was nevertheless possible to carry outthe time-resolved measurement of the switching process at a speed andwith a robustness comparable with that of the frequency response methoddescribed above.

While best analysis results can be obtained when recording the signalsduring both steps (A) and (B) it is nevertheless possible to base theanalysis on the signals of one of the steps only. This is particularlytrue for step (B), as experiments by the inventors have confirmed thatvaluable information about the bound target molecules can be discernedfrom a time-resolved analysis of the rising process of the probemolecule.

According to the first aspect of the invention, the apparatus furthercomprises an analysis module for automatically analyzing and/orprocessing the combined signal such as to determine the one or morecharacteristics of the target molecule. By integrating such analysismodule with the apparatus, the information of interest to the user ofthe apparatus can be provided rather than the experimental data itself,and this information or analysis result can preferably be presented tothe user by the output device.

In a preferred embodiment, the analysis module is configured to analyzeand/or process the combined signals such as to determine a time delaybetween

1. switching the external field between steps (A) and (B) and

2. the time-dependent signal reaching a predetermined threshold value.

Herein, the predetermined threshold value may for example correspond toa predetermined percentage of the maximum of the combined value. In thisembodiment, the analysis thus yields only two numeric values that can becorrelated with the size or effective Stokes radius of the targetmolecule bound to the probe molecule.

Note that even in this simple embodiment, the apparatus of the inventionyields more information than the frequency response method as describedabove, which only yields a single numeric value—the cut-offfrequency—reflecting both the dynamics of the transition from standingto lying (down transition) and from lying to standing (up transition).

At first sight one would assume that not much insight could be gained bythis additional information. After all, one would assume that thehydrostatic drag of the target molecule would have a similar effectduring up and down transitions, thus retarding both processes in asimilar way. This is true in some scenarios, and this is why thefrequency response method proved so successful in many cases. However,the inventors have observed that there are also scenarios in which thetarget molecule influences the up and down transitions in a dissimilarway. In particular, while of course the hydrostatic drag will alwaysplay a role in the switching dynamics, in some cases it may not play theonly and not even the decisive role. Instead, the inventors haveobserved that especially for the down transition, the dynamics also hasa stochastic component, which presumably has to do with the electricfield being confined to the electrode proximity due to theGouy-Chapman-Stern screening layer. It is believed that the probemolecule needs to fluctuate due to Brownian motion to a “startingconfiguration” before the external electric field can effectivelyinitiate the down transition. Accordingly, there is a time component tothe down transition unrelated or at least not directly related to thehydrodynamic resistance. Since the cut-off frequency determined with thefrequency response method always reflects a combination of up and downtransition time constants, the time constant of the down transition maydominate the result to an extent that smaller differences in theeffective Stokes radius may remain unobserved. This will be demonstratedbelow with reference to an actual example.

In addition or alternatively, the analysis module may be configured toanalyze and/or process the combined signals such as to determine thetime-derivative thereof. In particular, it is possible to determine onlythe maximum time derivative of the combined signal, which would as wellonly give a single number for the up and down transitions, respectively.However, it is believed that this number is more closely and directlycorrelated with the effective Stokes radius than e.g. the cut-offfrequency of the frequency response measurement. In particular, it isbelieved that the maximum signal derivative is largely unrelated to thestochastic delay of the transition due to Brownian motion, as itreflects the maximum speed of the transition, which is presumablygoverned by the effective Stokes radius.

In addition or alternatively, the analysis module may be configured tocompare the combined signal with empirical data or model data obtainedfrom an analytical model or a simulation. Experiments of the inventorshave shown that the time-resolved signals bear additional informationthat cannot be summarized in a single number. Instead, it appears thatthe signal versus time graphs for different targets have peculiar shapesand characteristic features that can be employed to identify targetswith more precision, for instance, with respect to the target moleculeshape or conformational flexibility. For example, it has been observedthat the graph of the signal versus time for some targets displays somecharacteristic kinks, while for other targets it is entirely smooth.Even if this behaviour is not entirely understood yet, this observationcan already be used to compare the recorded signal with empiricallyknown targets and to detect a match. This comparison can be automatizedand integrated into the analysis module.

Further, the inventors have also elaborated analytical models predictingthe signal to be expected for target molecules. An article “AnalyticalModel Describing the Molecular Dynamics of DNA-Protein ConjugatesTethered to Electrified Surfaces” by Andreas Langer, Wolfgang Kaiser andUlrich Rant will be submitted for publication shortly after filing ofthe present application. In this work, an analytical model describingthe switching behaviour of short double-stranded DNA molecules iselaborated, and model parameters are discerned from experimental data.After such a model is established, the time-resolved signal to beexpected for any given target size can be calculated, and the result ofthis calculation can be compared with the combined signal obtained withthe apparatus. This way it can for example be confirmed whether theexperimental data and the effective Stokes radius discerned therefrom isconsistent with the model or not. Accordingly, the analysis module mayoutput a reliability value together with the outputted characteristic ofthe target molecule.

The inventors have noticed that the switching process of the probemolecule is a stochastic process that is governed by Brownian motiontype effects plus a drift due to the external electric field. Morespecifically, it has been found that the switching dynamics can bedescribed very realistically based on a probability distributionp({right arrow over (x)},t) defining the probability that the probemolecule acquires a configuration {right arrow over (x)} at a time t ina time-dependent external field. In this model, the Stokes radius orsize of the target molecule is accounted for by a drift and/or adiffusion of the probability with regard to {right arrow over (x)}.Herein, {right arrow over (x)} can be any one- or more-dimensionalcoordinate that can parameterize the configuration of the probemolecule. The inventors have found that for a suitably stiff probemolecule, such as double-stranded DNA, the configuration can besufficiently parameterized by the angle α of the probe molecule withregard to the substrate.

In a preferred embodiment, the analysis module of the apparatus can beconfigured to determine a diffusion coefficient or a drift coefficientby fitting the solution for p({right arrow over (x)}, t) of aFokker-Planck equation containing said drift and/or diffusioncoefficient with the combined time-resolved signal, and configured toderive the size and/or Stokes radius of the target molecule from thedetermined drift and/or diffusion coefficient. Note that throughout thisdisclosure, the “combined time-resolved signal” refers to the averagetime-resolved signal that is generated from a large number ofconsecutive switching steps as referred to in steps (A) and (B) above.

It is seen that this rather simple model allows to already capture theessential physics behind the switching step and that it can be used todetermine the size and/or Stokes radius of the target molecule from theexperimental data with great precision. This will be furtherdemonstrated with reference to a specific example below.

In a preferred embodiment, the analysis module is configured to evaluatethe effective Stokes radius, the size and/or the molecular weight of thetarget molecule. In addition or alternatively to the above, the analysismodule may also be configured to evaluate the shape of the targetmolecule, in particular the folding state and/or a deviation from aglobular structure. A deviation from a globular structure can forexample be detected by a deviation of the signal from predictions of ananalytical model or simulation data based on a globular target molecule.

Further, the analysis module may be configured to evaluate or detect theaddition of further molecules to the target molecule.

Since the time resolved switching dynamics will depend on thetemperature and the chemical environment of a fluid environment of theprobe molecules, the combined signal can also be employed to determinechanges in these characteristics of the environment. Conversely, theinfluence of the environment, such as temperature or pH, on theproperties of the target molecules can also be determined, for instancethe temperature induced unfolding of a protein.

In a preferred embodiment, the electric field generating means of theapparatus comprises a wave form generator configured to generate asquare wave signal switching between a first and a second polarity.Herein, the period of the first and/or second polarity is chosen longenough such that the probe molecules can acquire the respective statesof maximum and minimum distance between the second portion and thesubstrate, respectively. Note that this is just the opposite of theprior art frequency response method, where the target moleculecharacteristics are revealed by the behaviour at a frequency where theprobe molecule can no longer follow the external AC field. Also, in thefrequency response method, a sine wave field is used rather than asquare wave. It should be noted that while in the setup of FIG. 1, aslow square wave potential is applied to the work electrode 16, in thiscase only the amplitude in the two polarization states is measured, butthere is of course no time resolved measurement of the up and downtransition between these states.

Preferably, the period of the first and/or second polarity of the squarewave signal is at least 1 μs, preferably at least 10 μs.

Preferably, the control means are adapted to repeat steps (A) and (B) atleast 10 times, preferably between 10³ to 10⁷ times before the recordedsignals are combined. Hence, in a preferred embodiment, it is possibleto record time-resolved measurements of a million up and downtransitions during only about 1 min, thus allowing to obtain goodquality averaged data in a short time, making the apparatus and methodespecially attractive for routine applications in research or industrylaboratories.

In a preferred embodiment, the detecting means of the apparatuscomprises a detector for detecting single photons emitted from afluorescence marker and means for determining the time delay or intervalbetween the switching of said external electric field between steps (A)and (B) and the detected photon. Herein, the control means is furtherconfigured to record each time interval in a histogram.

This embodiment is based on the observation that in view of the ratherfast switching times, the probability of registering more than onephoton or a few photons per up or down transition is not very high, thusallowing for a time-resolved measurement based on single photons. Evenif there are more than one photon during one transition, they can bedetected individually with a suitable circuitry allowing to measuremultiple photon events after a single trigger. The only limitation inthis regard is the so-called dead-time of the circuitry, i.e. the periodof inactivity of the circuitry after a photon event is detected. Sincethousands or even a million of up and down transitions can be measured,the resulting histogram will still record sufficient events to reliablyreflect the time-resolved transition dynamics.

In a preferred embodiment, the detecting means may comprise aramp-generator operatively coupled with the electric field generatingmeans such as to receive the switching of the electric field betweensteps (A) and (B) as a first trigger signal causing the ramp-generatorto start building up a voltage. The ramp-generator is also operativelycoupled with the detector such as to receive the detection of a photonas a second trigger stopping the voltage build up, wherein the built upvoltage is at least approximately proportional to the time differencebetween the two triggers.

Note that such a setup is generally known from so-called time-correlatedsingle photon counting (TCSPC) applied in fluorescence measurements,where the first trigger would be the excitation laser pulse and thesecond trigger would typically be the reception of a fluorescencephoton, and where the time delays between excitation and fluorescencewould be on the order of a few ns only. However, the inventors confirmedthat the TCSPC concept can also be very advantageously applied in thesurface-base molecular dynamics measurement of the invention. Inparticular, while the earlier time-resolved measurements of the inventorfor DNA only, i.e. without a capturing probe or a binding of targetmolecules, would not have suggested that such time-resolved measurementswould be feasible for a quick and routine measurement, employing thisTCSPC technique in fact allows for a very robust and reliableimplementation and short analysis times.

In an alternative embodiment, the detecting means may comprise anamplifier for amplifying the analogue signal indicative of the distanceof a second portion of the probe molecule from the substrate. If thesignal is the fluorescence signal of a fluorescence marker, then theamplifier could be an amplifier amplifying the signal of a photo sensor.Further, the apparatus may comprise means for recording and storing thetime-dependent amplified signal, in particular a digital storageoscilloscope (DSO) type device. The recording and storing means isoperationally coupled with the electric field generating means to betriggered to record the time-dependent signal by the switching of theexternal field between steps (A) and (B), and the recording and storingmeans is configured to combine the time-dependent signals to generate anaverage time-resolved signal.

The inventors have confirmed that alternatively to the TCSPC-method, thetime-resolved signal can be measured in a series of analoguemeasurements using a digital storage oscilloscope (DSO) type device. Theterm “DSO type device” indicates that in principle an ordinary DSO couldbe used, although of course the display functionality of the DSO is notneeded. Instead, all that is needed is the DSO's capability of atriggered recording of a time-dependent signal and the storing thereof.

The individual signals recorded are then added up to give a combinedaverage signal. Again, by superimposing thousands or a million signals,a combined signal of sufficient quality for a meaningful analysis can beobtained. The analogue detection approach also allows for a robust setupand short detection times.

As mentioned before, according to a second aspect, a method forevaluating one or more characteristics of a target molecule bound to aprobe molecule is provided. Alternatively or in addition to the above,the apparatus of the invention may comprise means for carrying out amethod according to any of the method claims enclosed herewith.

While the apparatus and method described above are specifically devisedfor a time-resolved measurement of the molecule switching dynamics assuch, it is also possible to determine one or more of the followingparameters from a plurality of combined signals obtained at differenttimes and/or at different concentrations of target molecules: a bindingrate and/or dissociation rate between the target and probe molecules, anaffinity constant and a dissociation constant. In these measurements, aparameter indicative of the switching dynamics may be continuouslysampled in short time intervals (e.g. 1 second) and monitored over time.Examples for such “switching dynamics” parameter are the integrated areaunder time-resolved curves (cf. FIGS. 8 & 9), or the real-timecalculation of the time-derivative of the “stand-up” process from thetime-resolved curves. Changes in these switching dynamics parametersover time are indicative of the binding/unbinding of target molecules ofthe probe layer in real-time. The data can be analyzed with standardmodels describing the binding kinetics of bi-molecular system.Association/dissociation rate constants as well as affinity constantscan be inferred from such an analysis.

According to a further aspect of the invention, the charge of the targetmolecule can be determined based on a measurement and an analysis of thedependency of the signal indicative of the distance of the secondportion of the probe molecule from the substrate on a static externalfield. The dependency of the signal on the static external field isreferred to herein as the “voltage response”. As will be demonstratedbelow, it turns out that the voltage response is a very sensitive toolto determine the charge of a target molecule bound to a probe molecule.The proposal of the “voltage response” method is influenced by thebetter understanding of the stochastic nature of the processes describedabove. In particular, depending on the charge of the target molecule,the voltage response curve will deviate in a characteristic way from thevoltage response curve of the free probe molecule alone. Accordingly, byobserving the deviation of the voltage response curve when a targetmolecule is bound from the voltage response curve of the free probemolecule alone, the polarity and even the size of the charge of thetarget can be determined.

Needless to say, an independent assessment of the target charge by meansof the “voltage response method” yields very important additionalinformation that will assist in an educated analysis of thetime-resolved data described above. Note that this aspect of theinvention does not rely on time-resolved measurements, and it can hencebe employed independently of the time-resolved measurement of theswitching dynamics as described above. Accordingly, this aspect can alsobe combined with prior art switch SENSE schemes, including schemes basedon an analysis of the frequency response of the switching dynamics, ashave been explained with reference to FIG. 2 above.

Consequently, according to a further aspect of the invention, anapparatus is provided which allows to determine the charge of a targetmolecule, said apparatus comprising: means for receiving a biochip, saidbiochip comprising a substrate to which probe molecules are attachedwith a first portion thereof, said probe molecules being charged andhaving a marker for allowing to generate signals indicative of thedistance of a second portion of said probe molecule from said substrate,said probe molecule being adapted to bind said target molecule, meansfor detecting said signal generated with said marker, means forgenerating an external electric field which said probe molecules areexposed to when said biochip is received in said receiving means, acontrol means configured to control the electric field generating meansto apply a sequence of static external fields with different fieldstrengths, wherein the control means is further configured to controlthe signal detecting means to record said signal indicative of saiddistance from said substrate as the function of external field strength,or—in other words—the voltage response curve, and an analysis module foranalyzing the recorded signals and to determine the charge of the targetmolecule based thereon. Herein, as mentioned before, the analysis mayinclude comparing the measured voltage response curve with the voltageresponse curve of the free probe molecule, i.e. without a targetmolecule bound thereto. This further aspect also relates to acorresponding method of determining the charge of a target moleculebased on the voltage response curve.

However, the apparatus described above which is adapted for carrying outthe time-resolved measurements, generally has all the prerequisites ittakes to record the voltage response curves. Accordingly, in a preferredembodiment, voltage response curves are recorded in addition to thetime-resolved measurement of the switching dynamics, and the result ofthe voltage response curves, i.e. information about the charge of thetarget molecule, can be accounted for in the analysis of thetime-resolved combined switching signals.

Based on the above functionalities, the apparatus and method of theinvention provide a very powerful analysis means. In particular, theapparatus and method allow to determine one or more of the following:

-   -   the presence of a certain target molecule in a sample,    -   the concentration of a target molecule in a sample,    -   the fraction of probe molecules occupied by a given target        molecule, or    -   the stoichiometric ratio of different target molecules that can        bind to the same probe molecule capture part, or of the same        target molecules in different configurations.

Herein, the term “target molecule” (singular) refers to the species oftarget molecules. It is understood that always a plurality of targetmolecules of the same species will be detected.

For example, if only one given target molecule is present in a sample,the combined signal will have a characteristic shape indicative of atleast the charge and the Stokes radius of the target. By comparing thecombined signal with a predetermined signal for a given target, thepresence of a certain target molecule in a sample can be identified.

In some instances, in particular for lower concentrations of targetmolecules, only a fraction of the probe molecules will be occupied by agiven target molecule. In this case, both, the free probe molecules andthe occupied probe molecules will add to the combined signal. Since thecontributions to the signal add up linearly, the total signal will be asuperposition of the signals of the free probe molecules and those ofthe occupied probe molecules, where the coefficients of thesuperposition will depend on the fraction of probe molecules occupied bythe target molecules. For example, if the fraction of occupied probemolecules was 70%, in the superposition, the coefficient of the signalfor the occupied probe molecules would be 0.7 and the coefficient of thesignal for the free probe molecules would be 0.3. In practice, when acertain combined signal is measured, the corresponding coefficients ofthe superposition can be determined by a fitting process such that thesuperposition coincides best with the measured combined signal. Thisway, the fraction of occupied probe molecules can be determined. Sincethe fraction of occupied probe molecules is related to the concentrationof the target molecules, it can be used as a measure of theconcentration of the respective target molecule.

The same principle and line of reasoning can also be applied fordetermining the stoichiometric ratio of different target molecules thatcan bind to the same probe molecule capture part, or of the same targetmolecules in different configurations. Herein, different configurationscould for example be different folding states of a protein that willlead to a different Stokes radius and hence a different switchingdynamics, as will be explained in more detail below.

Note that since the different target molecules bind to the same receptoror capture part of the probe molecule, there is no affinity selectivityby which the stoichiometric ratio could be discerned otherwise. However,if the time-resolved signals for the different target molecules, or forthe different configurations of the same target molecule, are known,again the coefficients of a superposition of the corresponding signalscan be determined that fit the measured combined signal. Herein, the“coefficients of the superposition” directly reflect the stoichiometricratio.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 A-D is taken from prior art document 1 illustrating the conceptof detecting the presence of target molecules by the switchSENSE method.

FIG. 2 A-B is taken from prior art document 1 and shows the frequencyresponse of the switching dynamics of pristine DNA and DNA with IgG(sheep) bound to it.

FIG. 3 A-C is taken from prior art document 1 and shows normalizedcut-off frequencies for DNA with proteins of varying sizes bound to it.

FIG. 4 is a schematic diagram of an apparatus according to an embodimentof the invention based on single photon counting.

FIG. 5 is a schematic diagram of an apparatus according to an embodimentof the invention based on a measurement of analogue signals.

FIG. 6 shows the results of a time-resolved fluorescence measurement ofa pristine DNA layer.

FIG. 7 is a diagram showing the normalized rise-time change as afunction of target molecule concentration.

FIG. 8 shows normalized time-resolved fluorescence signals for pristineDNA and DNA where a complete IgG antibody or a Fab2 antibody fragmenthave been bound to biotin protein receptors.

FIG. 9 shows the time-resolved normalized fluorescence signal of a DNAmodified with a protein receptor of the DNA after binding of a 50 kDaprotein and after binding an IgG antibody to the protein.

FIG. 10 A-C shows a comparison of results obtained from frequencyresponse measurements according to prior art and time-resolvedmeasurements according to the invention.

FIG. 11 shows a schematic representation of a model for modelling adouble-stranded DNA probe molecule.

FIG. 12 shows a comparison of the voltage response curves of the freeDNA as calculated by the analytical model and as measured in experiment.

FIG. 13 shows a comparison of the measured voltage response curves of afree DNA probe molecule and the DNA with a streptavidin target boundthereto.

FIG. 14 shows a comparison of the measured voltage response curves of afree DNA probe molecule and the DNA with an avidin target bound thereto.

FIG. 15 shows a measurement of the time-resolved fluorescence of a freeDNA probe molecule and the DNA with a DHFR target attached thereto.

FIG. 16 shows the time-resolved fluorescence for the DNA and DNA plusDHFR of FIG. 15 as calculated based on the analytical model, where therotation diffusion coefficient has been obtained by fitting to theexperimental data.

FIG. 17A shows time-resolved fluorescence curves obtained for differentconcentrations of target molecules.

FIG. 17B is a graph illustrating how stoichiometric ratios of differenttarget molecules can be determined by determining a superposition ofknown time-dependent fluorescence signals for the individual targets.

FIG. 18A shows time-resolved fluorescence signals for DNA probemolecules with 100% IgG occupation, 100% Fab occupation and 50% IgG-50%Fab-occupation.

FIG. 18B shows the best fit of the superposition of the individual IgGand Fab time-dependent fluorescence signals to the measuredtime-dependent fluorescence signal.

FIG. 19 shows time-resolved normalized fluorescence signals of a DNAprobe molecule occupied by folded and unfolded Fab, respectively.

FIG. 20 shows the time-derivative of the normalized fluorescence signalof DNA alone, DNA with streptavidin bound thereto and DNA with avidinbound thereto.

FIG. 21 A-F shows measurements revealing the forward and backward ratesof the binding kinetics for different proteins.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the preferred embodimentsillustrated in the drawings and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of the invention is thereby intended, such alterations andfurther modifications in the illustrated apparatus and method, and suchfurther application of the principles of the invention as illustratedtherein being contemplated as would normally occur now or in the futureto one skilled in the art to which the invention relates.

In FIG. 4, a first embodiment of an apparatus 20 for evaluating one ormore characteristics of a target molecule is schematically shown.

As is shown in FIG. 4, the apparatus 20 comprises an electrochemicalcell 22 adapted to receive a biochip 24 which is immersed in a liquid.The liquid could for example be an aqueous solution, such as pH-bufferedelectrolyte solutions, or complex physiological media, such as bloodserum, cell lysate or the like. The biochip 24 comprises a gold workelectrode 16 to which probe molecules are attached with a first portionthereof in a way similar to the panel C of FIG. 1. In the shownembodiment, the probe molecules have a fluorescence marker such asfluorescence marker 12 as shown in panel C of FIG. 1 allowing togenerate signals indicative of the distance of the fluorescence markerfrom the gold electrode 16 due to fluorescence quenching, as describedin the introductory part of the specification. In addition, a counterelectrode 26 made from platinum is provided in the electrochemical cell.

The apparatus 20 further comprises a wave form generator 28 and a switchmatrix 29 for applying a time-dependent bias between the work electrode16 and the counter electrode 26.

As is further shown in FIG. 4, a microscope 30 is provided for receivingfluorescence light from the fluorescence marker. Also, a laser 32 isprovided for exciting the fluorescence marker 12.

As is further shown in FIG. 4, a photo multiplier tube (PMT) 34 iscoupled with the microscope 30. The PMT 34 is capable of detecting asingle photon and to output a signal via signal line 36 in response tothe single photon detection. It is to be understood that instead of aPMT, other photon counting detectors can be employed, such as anavalanche photo diode (APD) or the like.

The PMT 34 is coupled with the trigger device 38 via a signal line 36. Afurther trigger device 40 is provided which is operatively coupled witha wave form generator 28. Both trigger devices 38, 40 are connected witha time-to-amplitude converter (TAC) 42. The TAC 42 is a highly linearramp generator that is started by a signal from trigger device 40 andstopped by a signal from trigger device 38 and as a result outputs avoltage that is proportional to the time difference between the twosignals.

The output of TAC 42 is coupled with a histogramming device 44. Thehistogramming device 44 is in turn coupled with an analysis module 46comprising a processor 48 and storage means 50 for empirical data andstorage means 52 for modelling software. Finally, the output of theanalysis module 46 is connected with an output device 54, such as adisplay.

Next, the operation of apparatus 20 will be described.

The wave form generator 28 generates a square wave signal with a periodof for example 100 μs, switching from positive to negative polarity orvice versa every 50 μs. This square wave potential is applied betweenthe work electrode 16 and the counter electrode 26. In response to thissignal, probe molecules such as probe molecules 10 shown in FIG. 1C willswitch between the standing and lying configurations back and forth. Thetrigger device 40 is operatively coupled with the wave form generator 28and inputs a trigger signal to the TAC 42 each time the square wavesignal switches its polarity. The trigger signal from the trigger device40 causes the TAC 42 to build up a charge at a strictly linear rate.

During the switching of the probe molecules, the laser 32 excites thefluorescence markers such as markers 12 of FIG. 1C. Single photons ofthe fluorescence light are detected with the PMT 34. If a photon isdetected, the trigger device 38 will stop the charge build-up of the TAC42. Hence, the charge that is built up in the TAC 42 will correspond toa time between the flank of the square wave signal, i.e. initiation ofthe switching transition, and the photon detection.

If the square wave is applied for e.g. 100 s, one million up transitionsand one million down transitions will take place, and during each ofthese transitions photons will be detected. For each detected photon, acorresponding time value is obtained by the TAC 42, and each time valueis recorded in a histogram by the histogramming device 44. Inparticular, each time the histogramming device 44 receives the timevalue from the TAC 42, it increases a count for the corresponding timebin. As a result, the histogram represents a time-resolved fluorescencemeasurement, where the time resolution is only limited by the bin size.

An example of such a histogram is shown in FIG. 6, where the normalizedfluorescence intensity for a 72 base-pair DNA layer modified with aprotein receptor but without any target molecules bound thereto isshown.

The time-resolved fluorescence intensity is inputted into the analysismodule 46 where it is analyzed and processed such as to evaluatecharacteristics of target molecules bound to the probe molecules in away described in more detail below. The result of the analysis is thenoutputted by the output device 54.

In FIG. 5, an alternative embodiment 56 of an apparatus according to theinvention is shown. Like and similar components of the apparatus 56 areprovided with identical reference signs as in the apparatus 20 of FIG.4, and the description thereof is omitted. In the apparatus 56, insteadof a PMT 34, a photo sensor 58 is provided, which generates a photocurrent in response to receiving fluorescence light. The photo currentis amplified by a current amplifier 60 and fed into an oscilloscope 62,which is a digital storage oscilloscope (DSO). The DSO 62 is alsooperatively coupled with the wave form generator 28. The DSO 62 istriggered by the transition or flank of the square wave signal of thewave form generator 28. Starting from this transition, the oscilloscoperecords and stores the amplified current signal provided by the photosensor 58 and the current amplifier 60. In other words, in thisembodiment, the input to the DSO 62 is an analogue signal indicative ofthe fluorescence intensity, which is recorded as a function of time,namely the time from the last transition of the wave form.

Again thousands or a million of switching cycles are carried out, and ineach of these cycles, the fluorescence intensity is recorded as afunction of time. The signals are added up by the DSO 62 such as togenerate a combined signal representing an average time-resolvedfluorescence signal similar to the one shown in FIG. 6.

While a DSO 62 is employed in the setup of FIG. 5, it goes withoutsaying that the displaying function of the oscilloscope 62 is notneeded, as only the DSO's capability of recording and storingtime-dependent signals is employed.

The combined time-resolved fluorescence signal is then inputted into theanalysis module 46 which is identical with the analysis module 46 of theembodiment of FIG. 4.

The inventors have built and tested both the apparatus 20 of FIG. 4 andthe apparatus 56 of FIG. 5 and found that with such a setup, atime-resolved measurement of the up and down transition process can bemeasured in short times on the order of 1 min and with a combined signalquality based on thousands or millions of switching transitions thatwill allow a meaningful analysis of the time-resolved switchingdynamics.

Next, experimental results obtained with the apparatus 20 of FIG. 4 willbe discussed. In FIG. 7, a diagram of the rise-time change versus theconcentration of a Fab2 fragment of an antibiotin IgG is shown. Uponbinding of the Fab2 to the DNA probe molecule, the rise-time of theprobe molecule from the lying to the standing configuration increasesfrom 3 μs to 8 μs (see FIG. 8 below). By observing the rise-time changeat different concentrations, the dissociation constant K_(D) can bedetermined. At high target concentrations, a saturated rise-time value(8 μs) is observed, which stays constant when further increasing theconcentration of target molecules. This value is associated with a 100%coverage (saturation) of probe molecules with targets. The initial, i.e.short, rise-time of the bare probe layer is associated with 0% targetcoverage. Intermediate coverage values for varying target concentrationscan be calculated from the rise-time values of the 0% and 100%coverages, respectively, which yields a so-called titration curve asshown in FIG. 7. By fitting the law of mass action (Langmuir isotherm)to the titration curve, the K_(D) value is obtained.

In FIG. 8, the time-resolved normalized fluorescence for the uptransition is shown for the DNA with a protein receptor (biotin)attached to the top (upper curve), for the DNA when a Fab2 antibodyfragment of a molecular weight of 100 kDa is bound to the biotin proteinreceptor (middle curve), and for a case where the complete IgG antibodywith a molecular weight of 150 kDa is bound to the biotin proteinreceptor (lower curve). Also shown in FIG. 8 is the charge of the workelectrode as a function of time, showing that the charge does notcorrespond to a true step function but also suffers from some finitetime constant.

As can be seen from FIG. 8, the various target molecules as bound to theprobe molecule can be clearly distinguished from each other and bedistinguished from a state where no protein is bound at all. Accordingto a preferred embodiment of the invention, the time-resolvedmeasurements are automatically analyzed by the analysis module 46 suchas to determine characteristics of the target molecule bound to theprobe molecule. For example, the analysis module 46 could determine thetime delay between the switching of the polarity of the external fieldand the time the normalized fluorescence signal reaches a predeterminedthreshold value, for example 50% thereof, which may be used to representthe rise-time of the probe molecule from the lying to the standingconfiguration. Such rise-time value is correlated with the size oreffective Stokes radius of the target molecule. Accordingly, theanalysis module 46 could determine an estimated effective Stokes radiusfrom the rise-time and output it via the output device 54.

Rather than determining the rise-time, for reasons given in the summaryof the invention, it may be preferable to determine the derivative ofthe normalized fluorescence which is indicative of the rising speed ofthe probe molecule and is expected to be a better indicator of theeffective Stokes radius. In particular, in a preferred embodiment themaximum of the time derivative of the normalized fluorescence can bedetermined, which is indicative of the maximum speed the probe moleculeacquires upon the up transition. Since it is believed that thehydrodynamic drag limits the maximum speed, the maximum speed will be amore direct measure of the hydrodynamic drag or effective Stokes radiusthan the rise-time, which may be influenced by other phenomena as well,including stochastic events.

However, it is also apparent from FIG. 8 that much more information iscontained in the time-resolved fluorescence, as it actually reflects theentire time-resolved dynamical behaviour. Accordingly, the analysismodule 46 may be configured for more sophisticated types of analysis,taking into account the stochastic nature of the electrically drivenprobe/target motion as described in the upcoming publication mentionedabove.

In one embodiment, empirical data for known targets are stored in astorage 50, and the analysis module 46 can automatically compare thetime-resolved fluorescence signal with empirical signals of knowntargets, thereby allowing to identify target molecules with greatercertainty. In cases like this, the analysis module may not only output acharacteristic of the target molecule, such as the effective Stokesradius, but can even identify the target molecule itself or output aconfidence value that the measured target molecule indeed coincides withthe assumed target molecule.

In addition or alternatively, the analysis module 46 may also comparethe measured time-dependent fluorescence of the up or down transitionwith data obtained from a model calculation, as has been explained inthe summary of the invention. Again, a comparison with a modelcalculation may help to identify a target molecule or at least to give aconfidence value that a certain target molecule identification or acharacteristic of the target molecule as presented by the analysismodule 46 is correct.

According to one embodiment, the probe molecule is a double-stranded DNAwhich is modelled as a charged rigid cylinder in which the charge iscontinuously distributed along the cylinder axis, as is schematicallyshown in FIG. 11. Accordingly, the configuration of the probe molecule10 can be parameterized by an angle α with regard to the substrate 16only. Note that the azimuth angle can be disregarded because it does notplay any role in the processes observed herein. According to the model,and with further reference to FIG. 11, the length of the DNA is measuredin multiples n of lengths b of a single base, where b=0.34 nm. Thediameter of the cylinder is 2R with R=1 nm. The charge q of the DNAdepends on the number of bases, i.e. q=−2ne, where e is the elementarycharge.

Since a potential is applied to the substrate 16, the DNA experiences anelectric field Φ(r, α), which decays exponentially:φ(r,α)=φ_(eff) ·e ^(−κT sin α)

Herein, Φ_(eff) is an effective potential that corresponds to theapplied potential Φ multiplied with a screening factor γ<1, i.e.φ_(eff)=γ·φ.

The inventors have found out that the dynamics of the probe molecule isto a large extent of stochastic nature. Accordingly, the motion of theprobe molecule can be described quite accurately based on Brownianmotion with an additional drift due to the applied electric field. Tofurther understand the dynamical behaviour of the probe molecule, theenergy U(α, Φ), the entropy S(α) and the Gibbs free energy G[α, Φ] forany given conformation, i.e. any given angle α is calculated as follows

${U\left( {\alpha,\phi} \right)} = {{{\gamma \cdot \phi \cdot \frac{q}{L}}{\int_{0}^{L}{e^{- {x{({{r\mspace{11mu}\sin\mspace{11mu}\alpha} + {R\mspace{11mu}\cos\mspace{11mu}\alpha}})}}}\ d\; r}}} = {{{- \gamma} \cdot \phi \cdot \frac{2e}{\kappa\; b} \cdot \frac{1 - e^{{- \lambda}\; L\mspace{11mu}\sin\;\alpha}}{{e^{x\; R\mspace{11mu}\cos\mspace{11mu}\alpha} \cdot \sin}\mspace{11mu}\alpha}} + U_{0}}}$S(α) = k_(b) ⋅ ln  Ω(α) = k_(b) ⋅ ln (N ⋅ 2π ⋅ L ⋅ cos   α) = k_(b) ⋅ ln (cos   α) + S₀${G\left\lbrack {a,\phi} \right\rbrack} = {{{U\left( {\alpha,\phi} \right)} - {T \cdot {S(\alpha)}}} = {{{- \gamma} \cdot \phi \cdot \frac{2e}{\kappa\; b} \cdot \frac{1 - e^{{- x}\; L\;\sin\;\alpha}}{{e^{x\; R\;\cos\;\alpha} \cdot \sin}\mspace{11mu}\alpha}} - {k_{b}{T \cdot {\ln\left( {\cos\mspace{11mu}\alpha} \right)}}} + G_{0}}}$

From this, the following Boltzmann probability distribution can bederived:

${{p\left\lbrack {\alpha,\phi} \right\rbrack} = {\frac{1}{Z} \cdot {\exp\left( {- \frac{G\left\lbrack {\alpha,\phi} \right\rbrack}{k_{b}T}} \right)}}},$with a normalization condition

${\int_{0}^{\frac{\pi}{2}}{{p\left\lbrack {\alpha,\phi} \right\rbrack}\ d\;\alpha}} = 1.$From the probability distribution, the fluorescence signal can then becalculated as follows:

${F\lbrack\phi\rbrack} = {\int_{0}^{\frac{\pi}{2}}{{{f\lbrack\alpha\rbrack} \cdot {p\left\lbrack {\alpha,\phi} \right\rbrack}}d\;\alpha}}$and${f\lbrack\alpha\rbrack} = {0.21 \cdot {\left( {1 - \left( \frac{{{L \cdot \sin}\;\alpha} + 1}{24} \right)^{- 2.8}} \right).}}$

Herein, f[α] is an analytical approximation of the height dependent dyefluorescence as described in the Journal of the American ChemicalSociety, 132, 7935 (2010).

Note that so far no time dependence has been introduced, since theelectric field has been kept stationary. However, with the aboveequation, it is possible to calculate the fluorescence signal fordifferent values of the applied static potential Φ. The correspondingcurve is referred to as “voltage response curve” in the following.

FIG. 12 shows a comparison of the voltage response curves as calculatedaccording to the above model and as obtained by experiment. In thecalculation, a screening factor of γ=0.018 has been assumed. Theagreement between the calculated voltage response according to the abovemodel and the measured data is excellent, which is a strong indicationthat the above model captures the essential physics correctly. Note thatthe screening factor γ can be determined by fitting the calculatedvoltage response curves to the measured curve.

Further note that the model so far only accounted for the free probemolecule, i.e. the double-stranded DNA, but not for any target molecule.As long as the stationary state is concerned, i.e. without atime-dependent electrical field, the target molecule will mainly affectthe results due to a possible charge thereof. In fact, based on theabove understanding of the stochastic behaviour of the probe molecule,the inventors conjectured that it should be possible to qualitativelyand quantitatively determine the charge of the target molecule from thevoltage response curve. This has actually been confirmed in experiment,as shown in FIGS. 13 and 14.

FIG. 13 shows the normalized fluorescence signal as a function of thestatic potential applied to the substrate 16, for both, the DNA alone(open diamonds) and the same DNA to which a negatively charged protein,namely streptavidin, was bound (filled squares). As can be seen fromFIG. 13, the negatively charged streptavidin obviously has a noticeableeffect on the voltage response curve in that the fluorescence signaldrops faster with increased substrate potential than in case of the DNAalone. This behaviour is intuitively understandable, since the negativecharge of the target molecule will add to the effect of the negativelycharged DNA, i.e. cause the probe molecule 10 to approach the substrate16 when a positive potential is applied thereto.

The opposite case is shown in FIG. 14, where a positively chargedprotein (avidin) is bound to the probe DNA. The corresponding voltageresponse curve is shown by filled circles, while the voltage responsecurve of the free DNA is again shown by open diamonds. It can be seenthat the voltage response curve in presence of the avidin also differsnoticeably from that of the DNA alone. The qualitative behaviour isagain intuitively understandable, as in this case the positively chargedtarget molecule is repelled from the positively charged substrate 16,which causes the voltage response curve to lie above that of the freeDNA for positive potentials.

Accordingly, it is seen that the voltage response curve is a verysensitive tool to determine the charge of a target molecule. Sincevoltage response curves can be recorded easily and quickly, this is thepreferred way of determining the charge of target molecules that can becarried out routinely in target molecule analysis.

Note that the charge Q of the target molecule can be easily introducedin the above model by introducing the following additional electricalinteraction term into the Gibbs-energy function:ΔU[α]=Q·φ _(eff) ·e ^(−κ·L sin α)

So far, the model has only accounted for stationary electrical fields.Once the electrical field Φ is time-dependent, the probabilitydistribution will be time-dependent too, i.e.φ(t)=+φ₀+Δφ·(1−e ^(+t/τ)), hence p[α,φ(t)]=p[α,t]

Assuming again that the dynamical behaviour of the probe molecule isstochastic in nature, the time dependency of the probabilitydistribution p(α, t) can be described by a Fokker-Planck equation:

$\frac{\partial{p\left\lbrack {\alpha,t} \right\rbrack}}{\partial t} = {{D_{r}\frac{\partial^{2}{p\left\lbrack {\alpha,t} \right\rbrack}}{\partial\alpha^{2}}} + {\frac{D_{r}}{k_{b}T}\frac{\partial\;}{\partial\alpha}\left( {\frac{\partial{G\left\lbrack {\alpha,t} \right\rbrack}}{\partial\alpha} \cdot {p\left\lbrack {\alpha,t} \right\rbrack}} \right)}}$

Herein, the term

$D_{r}\frac{d^{2}{p\left\lbrack {\alpha,t} \right\rbrack}}{\partial\alpha^{2}}$is a diffusion term characterizing Brownian motion like behaviour thatis governed by a rotational diffusion coefficient D_(r). The second termis a drift term due to the angle and time-dependent free energy. Theabove Fokker-Planck equation can be solved numerically for any giventime dependence of the electrical field Φ(t).

In order to simulate the DNA switching, one calculates the startingprobability distribution and then calculates the time evolution of theprobability distribution via the Fokker-Planck equation given above. Thesolution depends only on the rotational diffusion coefficient D_(r).Accordingly, D_(r) can be determined by fitting the model calculationsto the experimental data. This way, estimated rotational diffusioncoefficients of the free DNA and the DNA with the target molecule boundto its end can be determined. From this, one can in turn calculate thehydrodynamic radius of the attached target molecule using Stokes' law.

Again, it is seen that based on this model, the Stokes radius can bedetermined from the time-resolved signal with great precision. FIG. 15shows the time-resolved rising curves for bare double-stranded DNA andfor the same DNA with the enzyme dihydrofolate reductase (DHFR) bound toits end. As is clearly seen from FIG. 15, the rise time of the DNA withthe additional DHFR is retarded due to the friction caused thereby.

FIG. 16 shows the two best fits of solutions of the above Fokker-Planckequation, which were found for rotational diffusion radii of 0.12 μs⁻¹and 0.16⁻¹ μs⁻¹, respectively. From this, Stokes law yields ahydrodynamic radius of 1.6 nm for the DHFR, which almost exactly matchesthe literature value of 1.5 nm.

Accordingly, it is seen that the analytical model does not only help tounderstand the behaviour of the switching, but it can actually be usedto determine the Stokes radius of an unknown target molecule from thetime-resolved data with rather high precision.

Instead of analyzing the measured data with reference to an analyticmodel or a simulation, as mentioned before, experimental data can alsobe compared to stored data sets of known targets. Accordingly, bycomparison with known time-resolved data sets, unknown targets can becharacterized or even recognized.

FIG. 9 shows yet further examples of time-resolved normalizedfluorescence as obtained with the apparatus 20 of FIG. 4. In FIG. 9, theupper graph again shows the fluorescence of the time-resolved upwardswitching of a DNA modified with a protein receptor, but without anytarget molecule bound to it. The middle curve shows the time-resolvedfluorescence after binding of a 50 kDa protein to it. The lowest curveshows the time-resolved fluorescence after an IgG antibody (150 kDa) isbound to the 50 kDa protein. Again, FIG. 9 clearly shows that binding ofmultiple targets can be clearly distinguished with the time-resolvedfluorescence measurement of the invention.

Finally, with reference to FIG. 10, a comparison of the resultsobtainable with the prior art frequency response method and thetime-resolved measurement of the invention is shown. In FIG. 10a , thefrequency response curve of the pristine probe molecule in standardbuffer solution (black circles) and with an IgG antibody bound to it(white circles) is shown. The two curves can be clearly distinguished,and in particular, the effective stokes radius of the IgG antibody canbe evaluated by the shift of the cut-off frequency.

However, if the viscosity of the solution is increased by adding 50%glycerol to the fluid environment, the frequency response of the probemolecule with and without the IgG anti-biotin are identical.Accordingly, in this scenario, the anti-biotin binding to the probemolecule can no longer be distinguished.

FIGS. 10b and 10c show the time-resolved fluorescence measurements forthe same probe and targets for the up transition (FIG. 10b ) and thedown transition (FIG. 10c ).

As can be seen from FIG. 10b , even with 50% glycerol added to thesolution, in the up transition the probe molecule with and withoutanti-biotin can clearly be distinguished, yielding different rise-timesof 8.0 μs and 5.5 μs, respectively. However, it is seen that for bothbuffers, the time-resolved curves with and without anti-biotin in thedown transition can practically not be distinguished.

From the time-resolved measurements of FIG. 10b and FIG. 10c it is seenthat apparently, the hydrodynamic drag of the anti-biotin governs thedynamics of the up transition, but not of the down transition. This is aresult that could not be discerned from the frequency response analysisaccording to prior art.

What is more, the cut-off frequency will always be governed by both, thetime constants of the up and down transitions. In fact, the longer ofthe two time constants will dominate the cut-off frequency. The effectof this can be seen in FIG. 10a : Although the time constants for thedown transitions are nearly identical with or without anti-biotin boundto the probe molecule, the difference in the time constants of the uptransition (τ_(rise)) is sufficient to give rise to a shift in cut-offfrequency that allows to distinguish the two cases and to evencharacterize the anti-biotin with regard to its effective Stokes radius.However, when the viscosity of the buffer is increased by addingglycerol, although the rise-times with and without anti-biotin are stilldifferent, the cut-off frequency is dominated by considerably increasedtime constant τ_(fall) of the down transition to an extent that thefrequency response spectra can no longer be distinguished.

So in summary, FIG. 10 demonstrates a surprising and unforeseeableimprovement provided by the time resolved measurement of the inventionas compared to the frequency response analysis. While instrumentalexpenditure of the apparatuses 20 and 56 of FIGS. 4 and 5 is hardlyincreased as compared to an apparatus for carrying out the frequencyresponse analysis, it is a further and surprising result that thetime-resolved measurement can be put to practice in a very robust andreliable way and without significantly increasing the time for theanalysis. Given the more reliable results and the possibility for asophisticated analysis by analysis module 46, the time-resolvedmeasurement scheme of the invention is in fact particularly preferablefor apparatuses for routine use in laboratories, where detailed andreliable analysis results are to be provided without requiring the userto understand the underlying principle or interpret the measurementresults him- or herself.

The method and apparatus of the invention also allows to determine theconcentration of certain target molecules in a sample or thestoichiometric ratio of two or more target molecules in a sample. Thiswill be explained with reference to FIGS. 17 and 18.

FIG. 17(a) shows the time-resolved fluorescence signal for differentconcentrations of target molecules in a sample. The highest curve inFIG. 17(a) corresponds to a concentration of 0 pM, i.e. there are notarget molecules in the sample. Accordingly, this curve corresponds tothe switching behaviour of the probe molecule 10 alone.

The lowest of the curves are actually two curves that nearly coincideand correspond to target concentrations of 3 nM and 10 nM, respectively.As these two curves coincide, it can be assumed that the biosensor issaturated, i.e. that a target molecule is bound to each of the probemolecules 10. The two curves in-between correspond to intermediateconcentrations of 60 pM and 300 pM, and in this case obviously part ofthe probe molecules 10 are occupied by a target molecule while othersare not. Since the fluorescence signal is a linear combination ofindividual signals corresponding to probe molecules 10 with and probemolecules 10 without target molecules bound thereto, it is expected thatthe intermediate measured curves correspond to a superposition of thetarget-free curves and completely target binding curves. The respectivecoefficients of the superposition would then correspond to thepercentage of probe molecules with and without targets bound thereto.For example, if 80% of the probe molecules 10 are occupied by a targetmolecule, the resulting fluorescence signal curve is expected to be asuperposition of the lowermost (i.e. 100% binding) and uppermost (i.e.0% binding) curves in FIG. 17(a), where the coefficient of the lowermostcurve in the superposition would be 0.8 and where the coefficient of theuppermost curve the would be 0.2.

This conjecture is actually confirmed by experiments of the inventors.The inventors have prepared biochips with a plurality of probe molecules10, on which the receptor density, i.e. the density of capture portionswas varied, as is schematically shown in FIG. 17(b). For example, if thereceptor density was 50%, only half of the biomolecules 10 actually hada receptor for capturing a target molecule. Using a sample with a highconcentration of target molecules, it could then be ensured that allavailable receptors were actually occupied by a corresponding targetmolecule. Accordingly, by predetermining the receptor density,effectively the fraction of probe molecules with attached targetmolecules could be controlled.

In FIG. 17(b), the horizontal axis corresponds to the receptor density,i.e. the percentage of probe molecules 10 having a receptor. For each ofthese receptor densities, a time-dependent fluorescence signal as shownin FIG. 17(a) was recorded. Then the superposition of the known orexpected signal for free probe molecules and for 100%-targeted-probemolecules was determined that fitted the measured curve best. Thevertical axis of the diagram of FIG. 17(b) corresponds to thecoefficient of the free probe molecule curve in the respectivesuperposition. Accordingly, if the above hypothesis is correct, then alldata points should lie on the dashed line connecting the points (0,0)and (1,1) in the diagram of FIG. 17(b). As is seen from FIG. 17(b), thisis indeed the case, giving strong support that the hypothesis iscorrect.

So in summary, by knowing the 0% (i.e. free probe molecule) and the 100%coverage (i.e. completely targeted probe molecule) curves, the targetcoverage of any curve obtained from experiment can be determined withgood precision by determining the corresponding superpositioncoefficients of the 0%- and 100%-target-coverage-curves. Further, if itis known how the coverage relates to the concentration of targetmolecules in the probe solution, then this is a direct measure of theconcentration.

The same principle can of course not only be applied to determine thecoverage of receptors, but also to distinguish the ratio of differenttarget molecules that bind to the same receptor, in a sample solution.

For example, the stoichometric ratio of different target molecules thatcan bind to the same probe molecule receptor can be determined.According to prior art methods, this is hardly possible, since there isno affinity selection if both target molecules bind to the samereceptor. According to the invention, however, if the two differenttarget molecules lead to different time-resolved fluorescence curves,that are per se known, then in case of a measured curve (in the same wayas described above) a suitable superposition of the target-specificcurves can be determined that fits with the experimental time resolvedsignal, and the corresponding coefficients reflect the stoichometricratio. An example of this is shown in FIG. 18(a). In FIG. 18(a), thelower-most curve represents the time-dependent fluorescence of the IgGanti-biotin. The IgG can be fragmented, such that Fab-fragments areseparated from the IgG. The Fab-fragments can of course bind to the samereceptor (anti-gene) as the whole IgG. However, since the Stokes radiusof the Fab-fragment is smaller than that of the IgG, the time-resolvedfluorescence curve will rise quicker in the electrical field.Accordingly, the stoichometric ratio of IgG and Fab-fragments can bedetermined by the coefficients of a superposition of the known IgG-curveand the known Fab-curve that fits best with the experimental data.

This method has been confirmed in an experiment as well. However, inorder to be in a position to precisely pre-determine the stoichiometricratio of IgG and Fab, different receptors (anti-genes) have beenattached to the probe molecules 10. Half of the receptors were biotinwhich are receptors for the IgG anti-biotin, while the other half of thereceptors were digoxygenin, which were receptors of anti-digoxygeninFabs. The upper-most curve in FIG. 18(a) hence corresponds to the signalobtained for a 100% coverage of the anti-digoxygenin Fab.

Using a biochip with 50% biotin and 50% digoxygenin receptors, themiddle curve in FIG. 18(a) was measured. In practice, one would measurea curve like the middle curve in FIG. 18(a) and would then want to knowthe stoichiometric ratio of Fab and IgG. According to the teachingabove, one would look for the superposition of the known Fab- andIgG-curves that fit the measured data best. The result of this is shownin FIG. 18(b). The thick curve in FIG. 18(b) represents the actualfluorescence measurement, i.e. the middle curve in FIG. 18(a). The thinline in FIG. 18(b) is the best fit for the superposition of the upperand lower curves in FIG. 18(a) to the middle curve, which in the presentcase yielded 55% IgG and 45% Fab, i.e. superposition coefficients of0.55 and 0.45, respectively which is quite close to 50% IgG and 50% Fab.Accordingly, it is seen that the stoichiometric ratio can be determinedwith rather accurate position. This is a remarkable result, since in anactual application, there would be no affinity selection, i.e. thedifferent molecules would bind to the same receptors, and there is henceno other practical way of determining the stoichiometric ratio.

This embodiment of the invention will have many practical applications.For example, if an antibody like the IgG above shall be fragmented byadding an enzyme, the percentage of the fragmentization can bedetermined. Also, if a given molecule can form monomers or dimers, andthe time-dependent fluorescence curves for the monomer and the dimer,respectively, are known, then the stoichiometric ratio of the monomersand dimers in a sample can be readily determined.

In fact, with this embodiment of the invention, not only thestoichiometric ratio of different target molecules (like Fab/IgG ormonomer/dimer), but also the stoichiometric ratio of differentconfigurations of the same molecule can be determined, if the differentconfigurations lead to different time dependent fluorescent curves. Anexample for this is shown in FIG. 19. FIG. 19 again shows measurementsof the time-resolved normalized fluorescence. The upper-most curvecorresponds to the probe molecule 10 alone, which in this case again isdouble-stranded DNA. The lowermost curve corresponds to the probemolecule (DNA) to which a Fab fragment is bound. In the ordinary state,the Fab fragment acquires a “folded state” giving rise to a certainStokes radius that is responsible for the slower rise of thefluorescence signal as compared to the free DNA.

The middle curve in FIG. 19 corresponds to the same sample, to which,however, a detergent (SDS) is added. The SDS causes the Fab fragment tounfold, as is schematically shown in FIG. 19. In the unfoldedconfiguration, the effective Stokes radius is decreased, thereby leadingto a rise time that is between that of the DNA occupied by the foldedFab and that of the DNA alone. Accordingly, the change of theconformation, i.e. folded versus unfolded, can be directly observed bythe time-dependent fluorescence signal. After the SDS has been washedout of the solution, it was seen that the Fab fragments acquire theirfolded configuration again, i.e. the fluorescence signal of the lowestcurve in FIG. 19 was observed again. Also, the stoichiometric ratio offolded and unfolded Fabs can be determined by determining thecoefficients in the superposition in the same manner as described above.

As has been explained above, in many cases the time derivative of thefluorescence signal is a good observable for characterizing the targetmolecule. In FIG. 20, the time derivative of the fluorescence signal asa function of time is shown for the probe molecule (DNA) alone (dottedline), the probe molecule with a streptavidin target (dashed line) andthe probe molecule with a avidin target bound thereto (solid line).Avidin and streptavidin have practically identical size and Stokesradii, but still the time derivative of the fluorescence signal differsnoticeably. This difference is due to the charge of the target molecule.The streptavidin, which is negatively charged, will lead to a velocitythat remains higher than that of the (positively charged) avidin atleast in the second half of the stand-up process. This is intuitivelyunderstandable, since the negative streptavidin will support thenegative DNA in the stand-up motion, while the positively charged avidinwill counteract this motion. Again, this demonstrates that the methodaccording to the present invention is sensitive enough to evendistinguish the charge of target molecules from the time-resolvedfluorescence signal.

Finally, it is seen that the binding kinetics of the target molecules tothe receptor can be measured with very good precision. In FIG. 7, therise-time change with the concentration of a Fab2 fragment of anantibiotine IgG. was shown. From this, the dissociation rate K_(D) orits inverse, the affinity rate K_(A) can be determined. As iswell-known, the dissociation rate K_(D) corresponds to the ratio ofbackward rate (k_(off)) and forward rate (k_(on)),

${i.e.\mspace{14mu} K_{A}} = {\frac{1}{K_{D}} = {\frac{k_{on}}{k_{off}}.}}$However, in the framework of the present invention, it is also possibleto measure k_(on) and k_(off) directly. For this, in FIG. 21, themaximum value of the derivative of the normalized fluorescent signal,referred to as “V_(max)”, is shown after the probe molecules have beenexposed to the target (panels A, C and E) or after the exposure to thetarget molecules was terminated (panels B, D and F).

V_(max) is found to be a very sensitive indicator for analyzing whethera target molecule is bound to a probe molecule or not. As the probemolecules are exposed to the target molecules, the target molecules willbind to the probe molecules with the forward rate k_(on), therebyslowing down the switching dynamics and reducing V_(max). As is seen inpanels A, C and E, V_(max) decays exponentially as the probe moleculesare occupied by the target molecules with a rate that resembles k_(on).

Conversely, after the exposure to target molecules is terminated,V_(max) again increases according to 1−e^(−k) ^(off) ^(t)

FIG. 21 shows the binding kinetics for proteins A, G, L, INFα, MOG andFab, where the corresponding forward and backward rates k_(on), k_(off)are summarized in a table. Again, it is seen that the apparatus andmethod according to the present invention not only allow determining thedissociation or affinity rates K_(D), K_(A), respectively, but also theunderlying forward and backward rates k_(on), k_(off) with greatprecision.

Although preferred exemplary embodiments are shown and specified indetail in the drawings and the preceding specification, these should beviewed as purely exemplary and not as limiting the invention. It isnoted in this regard that only the preferred exemplary embodiments areshown and specified, and all variations and modifications should beprotected that presently or in the future lie within the scope of theappendant claims.

REFERENCE SIGNS

-   10 probe molecule-   12 marker-   14 protein binding tag-   16 work electrode-   18 biasing means-   20 apparatus for evaluating characteristics of target molecules-   22 receiving means-   24 biochip-   26 counter electrode-   28 wave form generator-   29 switch matrix-   30 microscope-   32 laser-   34 photo multiplier tube-   36 signal line-   38 trigger device-   40 trigger device-   42 time-amplitude-converter-   44 histogramming device-   46 analysis module-   48 processor-   50 storage for empirical data-   52 storage for modelling software-   54 output device-   56 apparatus for elevating characteristics of target molecules-   58 photo sensor-   60 current amplifier-   62 oscilloscope

What is claimed is:
 1. An apparatus for evaluating one or morecharacteristics of target molecules, said apparatus comprising: abiochip, said biochip comprising a substrate to which probe moleculesare attached with a first portion thereof, said probe molecules beingcharged and having a fluorescence marker for allowing to generatefluorescence signals indicative of the distance of a second portion ofsaid probe molecule from said substrate, said probe molecule beingadapted to bind said target molecule, means for generating and applyingan external electric field which said probe molecules are exposed to,said field generation and applying means comprising a wave formgenerator suitable for generating a square wave signal switching betweena first and a second polarity, detecting means for detecting saidfluorescence signal generated with said marker, said detecting meanscomprising: a microscope for receiving fluorescence light from thefluorescence marker, a single photon detector, coupled with saidmicroscope, configured for micro-second time scale detection of singlephotons emitted from a fluorescence marker, a first trigger coupled withthe single photon detector, a second trigger operatively coupled withsaid waveform generator, and a time-to-amplitude converter coupled withboth, the first and second trigger, wherein said first trigger isconfigured to input a first signal to the time-to-amplitude converter atthe time that the external electrical field switches its polarity, saidsecond trigger is configured to input a second signal to thetime-to-amplitude converter in response to a single photon beingdetected by said single photon detector, and wherein thetime-to-amplitude detector is configured to output a voltage signalrepresenting a time value corresponding to a time difference betweensaid first and second signals, and a histogramming circuit coupled to anoutput of said time-to-amplitude converter, said histogramming circuitconfigured for increasing, in response to receiving said time value, acount in a histogram time bin corresponding to said received time value,thereby generating a histogram representing a time-resolved fluorescencemeasurement, wherein the external electric field generated by saidwaveform generator causes: (A) the second portion of the probe moleculeto approach said substrate, and (B) the second portion of the probemolecule to move away from said substrate, while said signal detectingmeans detect single photons and update said time histogram to recordsaid fluorescence signal indicative of said distance from said substrateas a function of time during at least one of steps (A) and step (B), andwherein the electric field generation and applying means and thedetecting means are configured to repeat steps (A) and (B) for apredetermined number of times and to combine the recorded signals byupdating said histogram such as to generate an averaged time-resolvedsignal indicative of the process of said second part of said probemolecule approaching said substrate and/or moving away from saidsubstrate, said apparatus further comprising an analysis modulecomprising a processor, said analysis module configured for analyzingand/or processing said combined signal such as to determine said one ormore characteristics of said target molecule, and an output device or aninterface for directly or indirectly coupling an output device foroutputting the at least one or more characteristics of said targetmolecule.
 2. The apparatus of claim 1, wherein said analysis module isconfigured to analyse and/or process said combined signal to: determinea time delay between switching the external field between steps (A) and(B) and the time dependent signal reaching a predetermined thresholdvalue, wherein said predetermined threshold value preferably correspondsto a predetermined percentage of the maximum of the combined value,and/or determine the time-derivative of the combined signal, and/orcompare the combined signal with empirical data or model data obtainedfrom an analytical model.
 3. The apparatus according to claim 2, whereinsaid analytical model yields a probability distribution p({right arrowover (x)}, t) defining a probability that the probe molecule acquires aconfiguration {right arrow over (x)} at a time t in a time dependentexternal field, and the size and/or Stokes radius of a target moleculeis accounted for in said analytical model by a drift and/or a diffusionof the probability with regard to {right arrow over (x)}.
 4. Theapparatus according to claim 3, wherein said analysis module isconfigured to determine a diffusion coefficient or a drift coefficientby fitting a solution for p({right arrow over (x)}, t) of aFokker-Planck equation containing said drift and/or diffusioncoefficient with said combined time resolved signal, and configured toderive the size and/or Stokes radius of the target molecule from saiddetermined drift and/or diffusion coefficient.
 5. The apparatusaccording to claim 3, wherein the configuration is parameterized by anangle α of the probe molecule with regard to the substrate, and saiddiffusion coefficient is a rotational diffusion coefficient.
 6. Theapparatus of claim 1, wherein said analysis module is configured toevaluate one or more of the following target molecule characteristics:effective Stokes radius, size, molecular weight, the shape of the targetmolecule, in particular folding state and/or a deviation from a globularstructure, addition of further molecules to said target molecule, andthe charge of the target molecule.
 7. The apparatus of claim 1, whereinsaid analysis module is configured to determine temperature changes or achange in the chemical environment of a fluid environment of the probemolecules.
 8. The apparatus of claim 1, wherein said analysis module isconfigured to determine the effect of temperature changes or changes inthe chemical environment on the target molecule.
 9. The apparatusaccording to claim 1, wherein the first and/or second polarity has aperiod chosen long enough such that the probe molecules can acquire therespective states of maximum and minimum distance between said secondportion and said substrate.
 10. The apparatus according to claim 1,wherein the means for generating and applying an external electric fieldis configured to repeat steps (A) and (B) at least 10 times for acombined signal.
 11. The apparatus according to claim 1, wherein saiddetector comprises a ramp-generator operatively coupled with said meansfor generating and applying an external electric field, and configuredto receive the switching of the electric field between steps (A) and (B)as a first trigger signal causing the ramp-generator to start buildingup a voltage and operatively coupled with said single photon detectorsuch as to receive the detection of a photon as a second triggerstopping the voltage build up, said built up voltage being at leastapproximately proportional to the time delay between the two triggers.12. The apparatus according to claim 1, further configured to determinea forward rate (k_(on)) of the target molecule binding to the probemolecule and/or a backward rate (k_(off)) of the target molecule leavingthe probe molecule by observing how the maximum of the time derivativeof the combined signal changes in time after the probe molecules areexposed to said target molecules or after the exposure of said probemolecules to target molecules is terminated, respectively.
 13. Theapparatus according to claim 1, further configured to determine thecharge of said target molecule based on a measurement and an analysis ofthe dependency of said signal indicative of the distance of said secondportion of the probe molecule from said substrate on a static externalfield.
 14. The apparatus according to claim 1, further configured todetermine one or more of the following: the presence of a certain targetmolecule in a sample, the concentration of a target molecule in asample, the fraction of probe molecules occupied by a given targetmolecule, or the stoichiometric ratio of different target molecules thatcan bind to the same probe molecule capture part or of the same targetmolecules in different configurations, by carrying out the followingsteps: (A) exposing said sample to a biochip, said biochip comprising asubstrate to which probe molecules are attached with a first portionthereof, said probe molecules being charged and having a marker forallowing to generate signals indicative of the distance of a secondportion of said probe molecule from said substrate, said probe moleculecomprising a capture part capable of binding said target molecule oreach of said target molecules of said group of target molecules, (B)applying an external electric field causing the second portion of theprobe molecule to approach said substrate, (C) applying an externalfield causing the second portion of the probe molecule to move away fromsaid substrate, wherein during step (A) and/or step (B) said signalindicative of said distance of said second portion from said substrateis recorded as a function of time, (D) repeating steps (A) and (B) for apredetermined number of times and combining the recorded signals such asto generate an averaged time-resolved signal indicative of the processof said second part of said probe molecule approaching said substrateand/or moving away from said substrate, and (E) carrying out one of thefollowing steps: identifying the presence of a certain target moleculeby comparing said combined signal with a predetermined signal for saidtarget, or determining coefficients of a superposition of predeterminedsignals corresponding to the target-free probe molecule or the probemolecule with a respective target molecule bound thereto that fits thecombined signal.